title: High-throughput microfluidic single-cell digital polymerase chain reaction.
abstract: Here we present an integrated microfluidic device for the high-throughput digital polymerase chain reaction (dPCR) analysis of single cells. This device allows for the parallel processing of single cells and executes all steps of analysis, including cell capture, washing, lysis, reverse transcription, and dPCR analysis. The cDNA from each single cell is distributed into a dedicated dPCR array consisting of 1020 chambers, each having a volume of 25 pL, using surface-tension-based sample partitioning. The high density of this dPCR format (118,900 chambers/cm(2)) allows the analysis of 200 single cells per run, for a total of 204,000 PCR reactions using a device footprint of 10 cm(2). Experiments using RNA dilutions show this device achieves shot-noise-limited performance in quantifying single molecules, with a dynamic range of 10(4). We performed over 1200 single-cell measurements, demonstrating the use of this platform in the absolute quantification of both high- and low-abundance mRNA transcripts, as well as micro-RNAs that are not easily measured using alternative hybridization methods. We further apply the specificity and sensitivity of single-cell dPCR to performing measurements of RNA editing events in single cells. High-throughput dPCR provides a new tool in the arsenal of single-cell analysis methods, with a unique combination of speed, precision, sensitivity, and specificity. We anticipate this approach will enable new studies where high-performance single-cell measurements are essential, including the analysis of transcriptional noise, allelic imbalance, and RNA processing.
authors.affiliation: Centre for High Throughput Biology, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: A K
authors.initials: AK
authors.lastname: White
authors.forename: K A
authors.initials: KA
authors.lastname: Heyries
authors.forename: C
authors.initials: C
authors.lastname: Doolin
authors.forename: M
authors.initials: M
authors.lastname: Vaninsberghe
authors.forename: C L
authors.initials: CL
authors.lastname: Hansen
mesh_terms: D054736:Elongation Factor 2 Kinase / Q000235:genetics; D006801:Humans; D020014:K562 Cells; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D016133:Polymerase Chain Reaction / Q000295:instrumentation*; D017393:RNA Editing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis / Q000295:instrumentation*
chemical_list: D012333:RNA, Messenger; C517689:EEF2K protein, human; D054736:Elongation Factor 2 Kinase